Bacterial infections, especially infections caused by multi-drug resistant bacteria, pose a serious threat to human health and bring huge challenges to clinical treatment. The excessive use of antibiotics can easily lead to the emergence of bacterial resistance, which severely limits clinical treatment options. There is an urgent need to develop high-efficiency antibacterial materials and treatment strategies to inhibit infections caused by multidrug-resistant bacteria. In this work, a nanocomposite named Ofloxacin@HMPB@HA(OHH NPs) combined with the laser irradiation was used to reduce the development of drug resistance and accelerate wound healing in a model infected by Klebsiella pneumoniae(K.Pneumoniae). In vitro results showed that compared with OHH NPs or NIR laser irradiation alone, this combination strategy can exert a synergistic effect on anti-K.Pneumoniae by destroying cell integrity with generating ROS and reducing ATP, and also inhibit the development of bacterial resistance. Moreover, in vivo experiments have shown that the system effectively promotes wound healing through killing K.Pneumoniae and promoting the formation of new tissues. In summary, these results indicate that OHH NPs show great potential in the clinical application of bacterial infections.
Therapeutic proteins, including monoclonal antibodies, are typically manufactured using clonally-derived, stable host cell lines, since consistent and predictable cell culture performance is highly desirable. However, selecting and preparing banks of stable clones takes considerable time, which inevitably extends overall development timelines for new therapeutics by delaying the start of subsequent activities, such as the scale-up of manufacturing processes. In the context of the COVID-19 pandemic, with its intense pressure for accelerated development strategies, we used a novel transposon-based Leap-In Transposase® system to rapidly generate high-titer stable pools and then used them directly for large scale-manufacturing of an anti-SARS-CoV2 monoclonal antibody under cGMP. We performed the safety testing of our non-clonal cell bank, then used it to produce material at a 200L-scale for pre-clinical safety studies and formulation development work, and thereafter at 2000L scale for supply of material for a Phase 1 clinical trial. Testing demonstrated the comparability of critical product qualities between the two scales and, more importantly, that our final clinical trial product met all pre-set product quality specifications. The above expediated approach provided clinically-ready material within 4.5 months, in comparison to 12-14 months for production of clinical trial material via the conventional approach.
Generally, high bioelectroactivity of anodophilic biofilm favors high power generation of microbial fuel cell (MFC), however, it is not clear whether it can promote denitrification of MFC synchronously. In this study, the impact of anodophilic biofilms bioelectroactivity on denitrification behavior of single-chamber air-cathode MFC (SAMFC) in steady state was studied for the first time. Anodophilic biofilms of various bioelectroactivity were acclimated at conditions of open circuit (OC), Rext of 1000Ω and 20Ω (denoted as SAMFC-OC, SAMFC-1000Ω and SAMFC-20Ω, respectively) and run for 100 days in the presence of nitrate. Electrochemical tests and microbial analysis results showed that the anode of the SAMFC-20Ω delivered higher oxidation and denitrification current response and had a higher abundance of electroactive bacteria, like Geobacter, Pseudomonas and Comamonas, which possessed bidirectional electron transfer function, demonstrating a higher bioelectroactivity of the anodophilic biofilm. Moreover, these electroactive bacteria favored the accumulation of denitrifers, like Thauera and Alicycliphilus, probably by consuming trace oxygen through catalyzing oxygen reduction. The SAMFC-20Ω not only delivered a 61.7% higher power than the SAMFC-1000Ω, but also achieved a stable and high denitrification rate constant (kDN) of 1.9, which was 50% and 40% higher than that of the SAMFC-OC and SAMFC-1000Ω, respectively. It could be concluded that the high bioelectroactivity of the anodophilic biofilms not only favored high power generation of the SAMFC, but also promote the growth of denitrifers at the anodes and strengthened denitrification. This study provided an effective method and important theoretical basis for enhancing power generation and denitrification performance of the SAMFC synchronously.
Photosynthetic bacteria can be useful biotechnological tools – they produce a variety of valuable products, including high purity hydrogen, and can simultaneously treat recalcitrant wastewaters. However, while photobioreactors have been designed and modelled for photosynthetic algae and cyanobacteria, there has been less work on understanding the effect of light in photosynthetic bacterial fermentations. In order to design photobioreactors, and processes using these organisms, robust models of light penetration, utilisation and conversion are needed. This article uses experimental data from a tubular photobioreactor designed to focus in on light intensity effects, to model the effect of light intensity on the growth of Rhodopseudomonas palustris, a model photosynthetic bacterium. The work demonstrates that growth is controlled by light intensity, and that this organism does experience photoinhibition above 600 W/m2, which has implications for outdoor applications. Further, the work presents a model for light penetration in circular photobioreactors, which tends to be the most common geometry. The work extends the modelling tools for these organisms, and will allow for better photobioreactor design, and the integration of modelling tools in designing processes which use photosynthetic bacteria.
For drug products manufactured in mammalian cells, safety assurance practices are needed during production to assure that the final medicinal product is safe from the potential risk of viral contamination. Virus filters provide viral retention for a range of viruses through robust, size-based retention mechanism. Therefore, a viral filtration step is commonly utilized in a well-designed recombinant therapeutic protein purification process and is a key component in an overall strategy to minimize the risks of adventitious and endogenous viral particles during the manufacturing of biotechnology products. This review summarizes the history of viral filtration, currently available viral filters and prefilters, and viral filtration integrity test methods and study models. There is also discussion of current understanding and gaps with an eye toward future trends and emerging filtration technologies.
Chemically defined (CD) media are routinely used in the production of biologics in Chinese Hamster Ovary (CHO) cell culture and provide enhanced raw material control. Nutrient optimized CD media is an important path to increase cell growth and monoclonal antibody (mAb) productivity in recombinant CHO cell lines. However, nutrient optimization efforts for CD media typically rely on multi-factorial and experimental design of experiment (DoE) approaches or complex mathematical models of cellular metabolism or gene expression systems. Moreover, the majority of these efforts are aimed at amino acids since they constitute essential nutrients in CD media as they directly contribute to biomass and protein production. In this study, we demonstrate the utilization of multi-variate data analytics (MVDA) coupled with amino acid stoichiometric balances (SBs) to increased cell growth and mAb productivity in efforts to reduce CD media development efforts. SBs measure the difference between theoretical demand of amino acids and the empirically measured fluxes to identify metabolic states of the cell. When coupled with MVDA, the statistical models were not only able to highlight key amino acids towards cell growth or productivity, but also provided direction on metabolic favorability of the amino acid. Experimental validation of our approach resulted in a 55% increase in total cell growth and about an 80% increase in total mAb productivity. Increased specific consumption of stoichiometrically balanced amino acids and decreased specific consumption of glucose was also observed in optimized CD media suggesting favorable consumption of desired nutrients and a potential for energy redistribution towards increased cellular growth or mAb productivity.
The actinomycete Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. In previous studies the rebeccamycin production was significantly increased by the addition of glass beads during cultivation in different diameters between 0.5 – 2 mm and the induced mechanical stress by the glass beads was proposed to be responsible for the increased production. Thus, this study was conducted to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration and density (glass and ceramic) as well as shaking frequency, for a better understanding of the particle induced stress on L. aerocolonigenes. The induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency. In addition, the macroparticle-enhanced cultivation of L. aerocolonigenes was combined with soy lecithin addition to further increase the rebeccamycin concentration. With 100 g L-1 glass beads in a diameter of 969 µm and 5 g L-1 soy lecithin a concentration of 388 mg L 1 rebeccamycin was reached after 10 days of cultivation, which corresponds to the highest rebeccamycin concentrations achieved in shake flask cultivations of L. aerocolonigenes stated in literature so far.
Three dimensional printable formulation of self-standing and vascular-supportive structures using multi-materials suitable for organ engineering is of great importance and highly challengeable, but, it could advance the 3D printing scenario from printable shape to functional unit of human body. In this study, the authors report a 3D printable formulation of such self-standing and vascular-supportive structures using an in-house formulated multi-material combination of albumen/alginate/gelatin (A-SA-Gel)-based hydrogel. The rheological properties and relaxation behavior of hydrogels were analyzed prior to the printing process. The suitability of the hydrogel in 3D printing of various customizable and self-standing structures, including a human ear model, was examined by extrusion-based 3D printing. The structural, mechanical, and physicochemical properties of the printed scaffolds were studied systematically. Results supported the 3D printability of the formulated hydrogel with self-standing structures, which are customizable to a specific need. In vitro cell experiment showed that the formulated hydrogel has excellent biocompatibility and vascular supportive behavior with the extent of endothelial sprout formation when tested with human umbilical vein endothelial cells. In conclusion, the present study demonstrated the suitability of the extrusion-based 3D printing technique for manufacturing complex shapes and structures using multi-materials with high fidelity, which have great potential in organ engineering.
Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this work, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.
Microfluidic impedance cytometry is a powerful system to measure micro and nano-sized particles and is routinely used in point-of-care settings disease diagnostics and other biomedical applications. However, small objects near a sensor’s detection limit are plagued with relatively significant background noise and are difficult to identify for every case. While many data processing techniques can be utilized to reduce noise and improve signal quality, frequently they are still inadequate to push sensor detection limits. Here, we report the first demonstration of a novel signal averaging algorithm effective in noise reduction of microfluidic impedance cytometry data, improving enumeration accuracy and reducing detection limits. Our device uses a 22 μm tall microchannel and gold coplanar microelectrodes that generates an electric field, recording bipolar pulses from polystyrene microparticles flowing through the channel. In addition to outlining a modified moving signal averaging technique theoretically and with a model dataset, we also performed a compendium of characterization experiments including variations in flow rate, input voltage, and particle size. Multi-variate metrics from each experiment are compared including signal amplitude, pulse width, background noise, and signal-to-noise ratio (SNR). Incorporating our technique resulted in improved SNR and counting accuracy across all experiments conducted, and the limit of detection improved from 5 μm to 1 μm particles without modifying microchannel dimensions. Succeeding this, we envision implementing our modified moving average technique to develop next generation microfluidic impedance cytometry devices with an expanded dynamic range and improved enumeration accuracy. This can be exceedingly useful for many biomedical applications, such as infectious disease diagnostics where devices may enumerate larger-scale immune cells alongside sub-micron bacterium in the same sample.
Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the E. coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with non-natural amino acids incorporated at specific locations for producing homogeneous antibody drug conjugates. While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for non-natural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a five-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing non-natural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.
The realization of the enormous potential of stem cells requires development of efficient bioprocesses and optimization drawing drawn from mechanobiological considerations. Here, we emphasize the importance of mechanotransduction as one of the governing principles of stem cell bioprocesses, underscoring the need to further explore the behavioral mechanisms involved in sensing mechanical cues and coordinating transcriptional responses. We identify the sources of the intrinsic, extrinsic, and external noise in bioprocess under uncertainty, and discuss criteria and indicators that might assess and predict cell-to-cell variability resulting from environmental fluctuations. Specifically, we propose a conceptual framework to explain the impact of mechanical forces within cellular environment and identify key cell state determinants in bioprocess and discuss their implementation challenges.
In this work, we applied online chlorophyll a fluorescence measurements to monitor the changes in the photochemical parameters both in nitrate-deplete and nitrate-replete cultures of Nannochloropsis oceanica, in addition to biochemical parameters such as growth, lipid, fatty acid, and pigment contents. Under nitrate-replete conditions, growth was promoted along with pigment content, while total lipid content and fatty acid saturation level diminished. Under nitrate-deplete conditions, cultures showed an increased de-epoxidation state of the xanthophyll cycle pigments. Fast transients revealed a poor processing efficiency for electron transfer beyond QA, which was in line with the low electron transport rate due to nitrate depletion. Lipid content and the de-epoxidation state were the first biochemical parameters triggered by the change in nutrient status, which coincided with a 20% drop in the online effective quantum yield of PSII (ΔF/Fm’), and a raise in the Vj measurements. A good correlation was found between the changes in ΔF/Fm’ and lipid content (r=-0.96, p<0.01). The results confirm the reliability and applicability of online fluorescence measurements to monitor lipid induction in N. oceanica.
Molecular diagnosis is an essential means to detect pathogens. The portable nucleic acid detection chip has excellent prospects in places where medical resources are scarce, and it is also of research interest in the field of microfluidic chips. Here, the paper developed a new type of microfluidic chip for nucleic acid detection where stretching acts as the driving force. The sample entered the chip by applying capillary force. The strain valve was opened under the action of tensile force, and the spring pump generated the power to drive the fluid to flow to the detection chamber in a specific direction. The detection of COVID-19 was realized on the chip. The RT-LAMP amplification system was adopted to observe the liquid color in the detection chamber to decide whether the sample tested positive or negative qualitatively.
Adoptive cell immunotherapy with chimeric antigen receptor (CAR) T cell has brought a revolutionary means of treatment for aggressive diseases such as hematologic malignancies and solid tumors. Over the last decade, FDA approved three types of CAR-T cells against CD19 hematologic malignancies, including Tisagenlecleucel (Kymriah), Axicabtagene ciloleucel (Yescarta), and Brexucabtagene autoleucel (Tecartus). Despite outstanding results gained from different clinical trials, CAR-T cell therapy is not free from side effects and toxicities, and needs careful investigations and improvements. Gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system has emerged as a promising tool to address some of the CAR-T therapy hurdles. Using CRISPR/Cas9 technology, CAR expression as well as other cellular pathways can be modified in various ways to enhance CAR-T cell’s anti-tumor function and persistence in immunosuppressive tumor microenvironment. CRISPR/Cas9 technology can also be utilized to reduce CAR-T cells toxicity and side effects. Hereby, we discuss the practical challenges and hurdles related to the accuracy, efficiency, efficacy, safety and delivery of CRISPR/Cas9 technology to the genetically engineered-T cells. Combining of these two state-of-the-art technologies, CRISPR/Cas9 and CAR-T cells, the field of oncology has an extraordinary opportunity to enter a new era of immunotherapy, which offers novel therapeutic options for different types of tumors.
Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or product-specific challenges, such as low productivity or poor product quality. For other applications, systematic design of new media can facilitate the adoption of new industrially relevant alternative hosts. Despite manifold existing methods, common approaches for optimization often remain time and labor intensive. We present here a novel approach to conventional media blending that leverages stable, simple, concentrated stock solutions to enable rapid improvement of measurable phenotypes of interest. We applied this modular methodology to generate high-performing media for two phenotypes of interest: biomass accumulation and heterologous protein production, using high-throughput, milliliter-scale batch fermentations of Pichia pastoris as a model system. In addition to these examples, we also created a flexible open-source package for modular blending automation on a low-cost liquid handling system to facilitate wide use of this method. Our modular blending method enables rapid, flexible media development, requiring minimal labor investment and prior knowledge of the host organism, and should enable developing improved media for other hosts and phenotypes of interest.
Since 2014, an Asian lineage of Zika virus has caused outbreaks, and it has been associated with neurological disorders in adults and congenital defects in newborns. The resulting threat of the Zika virus to human health has prompted the development of new vaccines, which have yet to be approved for human use. Vaccines based on the attenuated or chemically inactivated virus will require large-scale production of the intact virus to meet potential global demands. Intact viruses are produced by infecting cultures of susceptible cells, a dynamic process that spans from hours to days and has yet to be optimized. Here, we infected Vero cells adhesively cultured in well-plates with two Zika virus strains: a recently isolated strain from the Asian lineage, and a cell-culture-adapted strain from the African lineage. At different time points post-infection, virus particles in the supernatant were quantified; further, microscopy images were used to quantify cell density and the proportion of cells expressing viral protein. These measurements were performed across multiple replicate samples of one-step infections every four hours over 60 hours and for multi-step infections every four to 24 hours over 144 hours, generating a rich dataset. For each set of data, mathematical models were developed to estimate parameters associated with cell infection and virus production. The African-lineage strain was found to produce a 14-fold higher yield than the Asian-lineage strain in one-step growth and a 7-fold higher titer in multi-step growth, suggesting a benefit of cell-culture adaptation for developing a vaccine strain. We found that image-based measurements were critical for discriminating among different models, and different parameters for the two strains could account for the experimentally observed differences. An exponential-distributed delay model performed best in accounting for multi-step infection of the Asian strain, and it highlighted the significant sensitivity of virus titer to the rate of viral degradation, with implications for optimization of vaccine production. More broadly, this work highlights how image-based measurements can contribute to discrimination of virus-culture models for the optimal production of inactivated and attenuated whole-virus vaccines.
The most effective way to prevent and control infectious disease outbreak is through vaccines. The increasing use of vaccines has elevated the need to establish new manufacturing strategies. One of the major approaches is cell-based production, which creates a need for high cell density to enable higher cell production levels. This has led to development of the technology of cell carriers, including micro and macro cell carriers. To follow the production process, quantifying the number of cells on these carriers is required, as well as the tracking of their viability and proliferation. However, owing to various carriers’ unique structures, tracking the cell’s is challenging using current traditional assays that were originally developed for monolayers of adherent cells. The current “gold standard” method is counting cell nuclei, separating cells from the carrier, staining with crystal violet and visually counting under a microscope. This method is tedious and counts both live and dead cells. A few other techniques were developed but were specific to the carrier type and involved specialized equipment. In this study, we describe a broadly ranging method for counting cells on carriers that was developed and employed as part of the production of a vaccine for use in the SARS-CoV-2 pandemic. The method is based on the Alamar blue dye, a well-known, common marker for cell activity, and was found to be successful in tracking cell adsorption, cell growth and viability on carriers. No separation of the cells from the carriers is needed, nor is any specialized equipment; the method is simple and rapid, and provides comprehensive details necessary for process control of viral vaccine production in cells. This method can be easily implemented in any of a number of cell-based processes and other unique platforms for measuring growth of encapsulated cells.